PROJECT SUMMARY Although immunostimulatory cytokines can be used to combat cancer, their poor stability and high off-target toxicity has limited their application in the clinic. The ?long-term goal of this project is to produce targeted anti-cancer cytokine mimetics with reduced toxicity. The ?overall objective is to apply recent breakthroughs in ?de novo protein design to yield a new category of targeted, non-toxic, immunostimulatory proteins. The ?central hypothesis is that the beneficial stimulatory effects of natural cytokines can be engineered into ?de novo designed proteins which do not engage in off-target binding, thereby circumventing the dose-limiting toxicities seen in clinical applications of natural/reengineered cytokines. The ?specific aims are to: (1) use ?de novo protein design to generate hyperstable, bioactive mimetics of interleukin-2, -4, -12, -15, and -21 which function by engaging with (i.e. binding to) interleukin receptors ?in vivo?; (2) to split these mimics into inactive parts which can regain activity by reassembling ?in vivo?; and (3) to fuse each of these inactive parts to specific targeting domains, thereby yielding conditionally-active cytokine mimics that stimulate T-cells by reassembling only on the surface of a targeted cells (i.e. cancer cells displaying two specific surface biomarkers). ?As proof of principle?, the first such ?de novo designed cytokine mimetic has been produced, split, and shown to reduce tumors in mice without accompanying toxicity or immunogenicity. This research proposal is ?innovative because it seeks to resolve a long-standing barrier to cancer immunotherapy (namely, the off-target toxicity of cytokine-based therapeutics) by designing from scratch a new class of non-toxic cytokine mimics. The proposal is ?significant because it would be the first example of computational protein design yielding targeted, biosuperior cancer therapeutics. Ultimately, such molecules have the potential to treat a wide range of cancers, including malignant melanoma, renal cell carcinoma, and more.